SITE-SPECIFIC THROMBIN RECEPTOR ANTIBODIES INHIBIT CA2+ SIGNALING ANDINCREASED ENDOTHELIAL PERMEABILITY

Thrombin receptor is activated by thrombin-mediated cleavage of the re ceptor's NH2 terminus between Arg-41 and Ser-42, generating a new NH2 terminus that functions as a ''tethered ligand'' by binding to sites o n the receptor. We prepared antibodies (Abs) directed against specific receptor domains to study the tethered ligand-receptor interactions r equired for signaling the increase in endothelial permeability to albu min. We used polyclonal Abs directed against the peptide sequences cor responding to the extracellular NH2 terminus [residues 70-99 (AbDD) an d 1-160 (AbEE)] and extracellular loops 1 and 2 [residues 161-178 (AbL 1) and 244-265 (AbL2)] of the seven-transmembrane thrombin receptor. R eceptor activation was determined by measuring changes in cytosolic Ca 2+ concentration ([Ca2+](i)) in human dermal microvascular endothelial cells (HMEC) loaded with Ca2+-sensitive fura 2-acetoxymethyl ester dy e. The transendothelial I-125-labeled albumin clearance rate (a measur e of endothelial permeability) was determined across the confluent HME C monolayers. AbEE (300 mu g/ml), directed against the entire extracel lular NH2-terminal extension, inhibited the thrombin-induced increases in [Ca2+](i) and the endothelial I-125-albumin clearance rate (>90% r eduction in both responses). AbDD (300 mu g/ml), directed against a se quence within the NH2-terminal extension, inhibited 70% of the thrombi n-induced increase in [Ca2+](i) and 60% of the increased I-125-albumin clearance rate. AbL2 (300 mu g/ml) inhibited these responses by 70 an d 80%, respectively. However, AbL1 (300 mu g/ml) had no effect on eith er response. We conclude that NH2-terminal extension and loop 2 are cr itical sites for thrombin receptor activation in endothelial cells and thus lead to increased [Ca2+](i) and transendothelial permeability to albumin.